Molecular cloning and characterization of allele d ‒ a newly identified allele of the 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) gene in apple - Short Communication

Abstract

Apple is a climacteric fruit of economic importance and high nutritional value. Better understanding of the apple ripening process is needed to improve fruit quality and extend apple shelf life. This paper presents the results of molecular cloning and characterization of a newly identified allele of the ripening-specific ACO1 gene from Malus floribunda 821. A polymorphism of the ACO1 gene was detected using the polymerase chain reaction and BamH1 and RsaI enzymatic digestion. Whilst cloning and sequencing data confirmed the identity of allele d, it also revealed high sequence conservation (97.3 to 98.9%). In addition, a comparison of sequence data revealed some differences within the coding regions of the ACO1 alleles identified so far.

Characterisation of indigenous apple accessions with respect to polymorphism of ACS1 and ACO1 genes

“…The amplification of the ACO1 gene by using M11 and M12 primers (Castiglione et al, 1999) resulted in a PCR product of approximately 1,600 bp (Figure 2a). The size of the PCR fragment was consistent with the ACO1 product reported for autochthonous and foreign cultivars (Marić et al, 2005a;Marić and Lukić, 2014), selections singled out at the Fruit Research Institute, Čačak (Marić et al, 2005b) and seedlings of an interspecific F1 progeny from the cross 'Fiesta' × 'Totem' (Fernández-Fernández et al, 2008) and Malus species (Marić, 2016;Marić et al, 2019). Given that a monomorphic PCR product was obtained, further identification of ACO1 alleles was based on its restriction analysis conducted by digestion with BamH1 [yielded a monomorphic fragment of 830 bp and a segregating fragment of 760 bp (f2); another segregating fragment of 700 bp (f1) was not observed] and RsaI [yielded a monomorphic fragment of 230 bp and two segregating fragments of 530 and 680 bp (considered as f1 fragment) and 1,270 bp (f2)] enzymes.…”

“…Therefore, among ACO1 alleles, A and B, firstly revealed by Castiglione et al (1999), correlate with the alleles a and b, reported by Marić and Lukić (2014), and the alleles ACO1-2 and ACO1-1, published by Costa et al (2005), as well as with the alleles c, d and n, found by Marić et al (2005b), Marić and Lukić (2014) and Marić (2016). To date, the allele d of the ACO1 gene has been confirmed only in nine wild Malus species (Marić, 2016;Marić et al, 2019).…”

“…Discrimination of ACO1 alleles is based on the amplification of the entire gene and subsequent digestion with restriction enzymes (Marić et al, 2005a,b;Marić and Lukić, 2014) or on the fragment amplification that encompasses part of the third and the fourth exons, including the third intron which shows a large length polymorphism (Costa et al, 2005). Therefore, among ACO1 alleles, A and B, firstly revealed by Castiglione et al (1999), correlate with the alleles a and b, reported by Marić and Lukić (2014), and the alleles ACO1-2 and ACO1-1, published by Costa et al (2005), as well as with the alleles c, d and n, found by Marić et al (2005b), Marić and Lukić (2014) and Marić (2016). To date, the allele d of the ACO1 gene has been confirmed only in nine wild Malus species (Marić, 2016;Marić et al, 2019).…”

“…The identification of ACO1 alleles in the current study was performed according to our previous studies, i.e. PCR amplification of the entire gene, which encompasses four exons and three introns, followed by digestion with two restriction enzymes (Marić et al, 2005a,b;Marić and Lukić, 2014;Marić, 2016;Marić et al, 2019). This manner made it possible to reveal four alleles (a, b, c and d) of the ripening-specific ACO1 gene, whose identity was confirmed by cloning and sequencing of their full-length nucleotide sequences (Marić and Lukić, 2014;Marić, 2016).…”

“…'Cox's Orange Pippin', 'Golden Delicious', 'Jonathan' and 'McIntosh' dominated mostly in earlier programmes, whilst 'Gala' and 'Fuji' are now the most commonly used parental cultivars (Noiton and Alspach, 1996;de Albuquerque Jr. et al, 2011). Apple breeding programmes need new germplasm, such as indigenous genotypes which appear to carry useful traits (Marić et al, 2016).…”